I thought I'd add more about physical attributes and your question in general. While NAD+ is important I just don't think it's going to be the end game. When it comes to physical attributes, especially appearances, these things are going to be better addressed by AGE breakers than anything else. We have yet to discover a means of removing the glucosepane that accumulates in the extra-cellular matrix. (ECM). I really think CR extends lifespan not just because it activates SIRT which feeds off NAD+, repairing DNA, etc, etc, but because more importantly, it greatly reduces the accumulation of AGEs. A calorie restriction mimetic like NR or any NAD+ booster isn't the whole picture or the whole equation. AGEs are a big part of the problem. I can't stress enough that if you're limited on funds and want to do something about aging, spend your money on these particular SENS projects rather than supplements. It's cheaper to just fast frequently, drink plenty of green tea and take carnosine. Also don't cook at such high temps. Anything that reduces formation of AGEs is a good thing.
So, there's a lot here, of course ... I'm going to address what seem to be the main points of confusion:
First, AH, thanks for engaging here: you're clearly highly knowledgeable in this area, and your input is welcome (as would be direct communication with us, particularly if you're engaged in intervention-oriented research on glucosepane, especially since you're doing so using an approach consistent with the "damage-repair" strategy of SENS).
It is of course correct that glucosepane is actually pretty labile: as you probably know, the reason it went undetected for so long was exactly that the harsh treatments and broad-spectrum proteases normally used to break down aging collagen to look for nonenzymatic crosslinks destroy it in the process. It was only identified through a painstaking process of sequential enzymatic digestion of the native collagen. This is one of the many reasons why having a ready source of synthetic glucosepane is valuable: by incorporating it into collagen peptides, it can be used to generate antibodies for the convenient labeling of glucosepane in vitro and in vivo.
A viable glucosepane-cleaving rejuvenation biotechnology, then, must to selectively cleave glucosepane crosslinks without damaging the native collagen. This would both directly restore its youthful elasticity, and should also allow for normal, physiological turnover of the tissue (such as by restoring MMP1's ability to access to its binding site, which the molecular dynamics simulation of glucosepane formation linked by Slicer suggests is likely impeded by glucosepane because its favorable formation site is only two amino acid residues away).
This is also the resolution of the conundrum highlighted by Slicer: [Since] MMPs, no matter the amount, cannot function on collagen with glucosepane crosslinks ... Can we develop something to finish what the MMPs started? Is there any molecule that can either usurp glucosepane's hold on its binding sites without damaging anything else or simply go around it, unmaking collagen in an entirely different way and breaking off the glucosepane-infested chunks?
Neatly cleaving glucosepane from the native structure will achieve this aim in a more direct and durable way than trying to unbind it or work around it, and (as noted) will not only allow for the turnover of the previously-crosslinked collagen fibril, but also restore its motion and function, likely rendering turnover unnecessary unless other damage has accumulated.
It's quite true that glucosepane accumulates much more slowly in normally-aging and even diabetic rats than it does in human aging. This isn't really an impediment to using them for basic testing of candidate glucosepane-cleaving agents, however. Even if glcosepane's low tissue burden in rats means that cleaving it will not have dramatic rejuvenating effects in these animals (which is a reasonable prediction, but might be happily disproven in the event), its high prevalence in aging and diabetic human collagen, and its implication in the complications of diabetes, will make the mere demonstration of a candidate's ability to cleave glucosepane crosslinks in vivo a sufficient proof-of-concept to spur further work to move it down the therapeutic pipeline into human testing.
(It's perhaps worth noting that even if glucosepane did accumulate in rats in a way that more closely scales to their rate of aging, cleaving it wouldn't necessarily extend their lifespan, because of the "weakest link in the chain" problem: to extend the lives of otherwise-healthy, normally-aging mice or humans by definition requires addressing all of the cellular and molecular damage driging aging to some degree, using a comprehensive panel of rejuvenation biotechnologies. As Chief Science Officer de Grey wrote to address a canard along these lines from critics:
[It is true] that no SENS intervention—in isolation—has ever been shown to extend any organism's lifespan. I do not recall Henry Ford alerting potential customers that the components of a car—in isolation—remain obstinately stationary when burning petrol is poured on them, nor do I recall his being castigated for this omission.
And in the meantime, the free availability of the anticipated glucosepane-detecting antibodies generated by the research that SENS Research Foundation funded at Yale will enable the identification of more suitable animal models for the demonstration of a hard health outcome. It's even conceivable (though not likely) that the mouse might be one such: the 300 pmol/mg figure cited by AH is for rats, and conceivably mice might prove a closer parallel to human aging and diabetes in this regard). On this front:
AH, you noted that Although glucosepane has recently been synthesized in vitro (I assume you mean by Draghici et al), I find it unlikely that work can have applications for animal models. Simply infusing glucosepane will not form the collagen crosslinks in question.
That wouldn't be the idea. The ability to synthesize glucosepane is now enabling the Yale crosslink team to incorporate glucosepane into synthetic, chemically-uniform crosslinked peptides. These can be used to create antigens with which to immunize rabbits, inducing the formation of antibodies targeting glucosepane-containing peptides. Labeled monoclonal antibodies generated from hybridomas derived from B-cells from such animals could then be used for the convenient detection of glucosepane crosslinks in tissue samples, and then evaluate the effects of candidate glucosepane-cleaving rejuvenation therapies in vitro and in vivo.
You (AH) also express concern that bacteria and other microorganisms just need to find something which works [to degrade glucosepane]. It's not their problem if the solution they use won't help us for the odd situation we're trying to solve."
Right ... the idea, however, is to find bacteria that can grow under physiologic conditions (temperature, pH, etc) with glucosepane as the sole available energy source, or alternatively as the sole available source of lysine and arginine for strains auxotrophic for these amino acids. In the wild, a community of bacteria and other microorganisms will often cooperate or compete for energy sources, leading to strains that are specialized in attacking specific bonds within a larger structure to carve out a niche for themselves (or a role within a larger network). It is then trivial to negatively screen candidate enzymes identified through such methods against physiologic collagen and later other proteins.
In addition to wild-derived bacteria, the Yale group is working to construct a cosmid library (see also here) to screen the huge diversity of gut microbe enzymes in easily-cultured E. coli lines, which would then similarly be screened against glucosepane as the sole source of energy and/or lysine and arginine.
Another issue with enzymes is the tight packing of the collagen fibrils, making it questionable if the enzyme will be able to reliably reach its target. A tailored small molecule may be a considerably better option for that reason alone.
Entirely possible, yes. The advantage of a microorganism-based enzyme-discovery program is that the enormous diversity of solutions generated by evolution (both in nature and later through directed enzyme evolution) allows for the hypothesis-neutral testing of an enormous range of candidate therapies generated by the engines of evolution, which are much cleverer than I am . At the moment, I don't think anyone has much of an idea on how to identify good small molecule candidates with conventional medicinal chemistry methods (though I'm intrigued to hear that you have some thoughts, AH!).
I'm a also going to quote from a recent email from Dr. de Grey, from a conversation on this subject in which I was a participant:
the access issue for enzymes reaching crosslinks in tightly-wound collagen is one that has been on our mind since forever, and is in tension with the converse problem of specificity of small molecules. The fact that glucosepane looks so weird, seven-membered ring etc, does not translate as well as I had initially hoped to the conclusion that drugs to cleave it will often be harmless to structures that the body synthesises on purpose. For this reason one approach that we are looking at is organocatalysts, which are small peptides with enzymatic activity - they are a compromise between size and specificity, and thus may be the best of both worlds.
But there is a huge amount not known about all this; it may just be that regular enzymes work fine because breaking the subset of glucosepane that they can get to will loosen things up enough that they can get to a bit more, and so on.
No matter what the strategy one favors for identifying therapeutic candidates, the glucosepane reagents generated by David Spiegel's group through the SENS Research Foundation funding will for the first time provide the soil that will allow a thousand flowers to bloom. Like you, AH, and I'm sure like everyone here, I'd be delighted to make progress toward a viable glucosepane-degrading therapeutic based on on any strategy, though it would sure be nice to claim a part in the bragging rights for that, too . Solvitur ambulando!